Fig. 8. Bcl-2 expression in MDCK cells decreases the cytoskeleton-associated pool of the junctional proteins and the surface E-cad. (A) Confluent, vector transfected (Neo) and two Zn²⁺-induced independent MDCK-Bcl-2 transfectants (B1, B2) were extracted with CSK buffer and the soluble (S) and insoluble (P) fractions separated. Fractions were resolved on 10% (Bcl-2 and tubulin) or 6% (E-cad, -cat, ß-cat, Pg, and ZO-1) gels and processed for IB with various antibodies. This experiment was repeated 5 times producing similar results with very little variability among the experiments. (B) The protein bands in A were scanned and quantitated using the NIH imager software. The value obtained for each protein/cell line was normalized to the value obtained for tubulin in the same lysate/cell line and the ratio of the soluble to insoluble calculated. (C) Confluent MDCK-Neo and Zn²⁺-induced MDCK-B1 and MDCK-B2 cultures were biotinylated and equivalent amounts of total proteins precipitated by streptavidinagarose beads. Biotinylated complexes were eluted, separated on SDS/6% gels and together with 50 µg of the TCE from each line were processed for IB with E-cad antibodies. To confirm equal loading, the TCE blots were reprobed with tubulin antibodies. (D) Confluent MDCK-Neo and Zn²⁺-induced MDCK-Bcl-2 cells were either fixed and then permeabilized (Total) or CSK-extracted and then fixed (Surface) and processed for staining with an E-cad antibody (3G8). Scale bar: 50 µm.

Return to article